Selective removal of cells having accumulated agents

ABSTRACT

A method of treating a condition associate with accumulation of an agent in cells in a patient includes exposing the cells to ultrasound, to selectively kill or induce apoptosis in the cells. The cells include the accumulated agent.

BACKGROUND

Accumulation of agents, such as proteins, lipids, bacteria, viruses,parasites or particles, leads to, or is associated with, pathologicalconditions. For example, nucleolin, a protein normally expressed in thenucleus or the cytoplasm has been shown to be expressed at the cellsurface in neoplastic cells and endothelial cells of angiogenic vesselsin vivo. Mi Y, et al. Apoptosis in leukemia cells is accompanied byalterations in the levels and localization of nucleolin. J Biol Chem278:8572-9 (2003); Sven C, et al. Nucleolin Expressed at the CellSurface is a Marker of Endothelial Cells in Angiogenic Blood Vessels,Journal of Cell Biology, Vol. 164, No. 4, 871-878 (2003). Anotherexample is P-glycoprotein (P-gp), a plasma membrane protein, which isover expressed in tumor cells that present a multidrug resistance (MDR)phenotype, which causes efflux of several structurally unrelatedtherapeutic drugs used for cancer treatment. Loo T W, et al.Identification of Residues in the Drug Translocation Pathway of theHuman Multidrug Resistance P-glycoprotein by Arginine Mutagenesis,Journal of Biological Chemistry, Vol. 284, No. 36, 24074-24087 (2009).Deposition and subsequent accumulation of intracellular proteinaggregates has been observed in several neurodegenerative disorders,such as α-synuclein in Parkinson's disease, β-amyloid and tau inAlzheimer's disease, and huntingtin in Huntington's diseases, and prionprotein (PrP) in transmissible prion encephalopathies. Brandin P, et al.Prion-Like Transmission of Protein Aggregates in NeurodegenerativeDiseases, Nat Rev Mol Cell Biol. Vol. 11, No. 4, 301-307 (2010). Poly Abinding protein (PABP) accumulates in the cytoplasm of betaherpesviruses (HCMV)-infected cells. Perez C, et al. Translationalcontrol of cytoplasmic poly A binding protein (PABP) abundance inHCMV-infected cells, J Virol. October 27 (2010) Epub.

Intracellular lipids accumulation is commonly observed in advancedatherosclerotic plaques. Monocyte infiltration in the intima layer ofthe vascular wall is followed by differentiation into macrophages, whichin turn take up modified lipoproteins and become macrophage foam cellsas a result of such intracellular lipids accumulation. Persson J, et al.Interleukin-1 beta and tumour necrosis factor-alpha impede neutral lipidturnover in macrophage-derived foam cells, BMC Immunology, 9(7) (2008).Obesity is associate with the accumulation of lipids in fat cells.

Some bacteria may accumulate inside cells, for example Mycobacteriumtuberculosis and Pseudomonas aeruginosa. M. tuberculosis causes theformation of hard nodules or tubercles in the lungs, parasitizesmacrophages by blocking the phagosome-lysosome fusion, a process calledphagosome maturation arrest, and by replicating inside the phagosome.Vergne I, et al. Cell Biology of Mycobacterium tubercolosis tuberculosisPhagosome, Ann Rev Cell Dev Biol., Vol. 20, 367-94 (2004). Similarly, P.aeruginosa colonizes the lungs of patients with cystic fibrosis andproduces biofilms, alginates, and specific lipid A modifications, whichallow the bacteria to escape immune response and cause severe chronicinflammation. Moskowitz S M, et al. The Role of PseudomonasLipopolysaccharide in Cystic Fibrosis Airway Infection, SubcellBiochem., Vol. 53, 241-53 (2010). Production of biofilms by Haemophilusinfluenzae, Streptococcus pneumoniae, and other bacteria, has beenlinked to chronic otitis media in pediatric patients. Hall-Stoodley L,et al. Direct Detection of Bacterial Biofilms on the Middle-Ear Mucosaof Children With Chronic Otitis Media, JAMA, Vol. 256, No. 2, 202-11(2006).

Some protozoan parasites present intracellular accumulation, for examplePlasmodium, Leishmania, Trypanosoma and Toxoplasma. Plasmodium, theagent causing malaria, replicates and accumulates inside erythrocytes,provoking cell rupture and dissemination of the agent, while the mainsites of sequestration of the infected erythrocytes containing thetrophozoites, schizonts and gametocytes of the parasite have been shownto be the lung, spleen, and adipose tissue, but also the brain, skin,bone marrow, and skeletal and cardiac muscle. Franke-Fayard B, et al.Sequestration and Tissue Accumulation of Human Malaria Parasites: Can WeLearn Anything from Rodent Models of Malaria?, PLoS Pathogens, Vol. 6,No. 9, e1001032 (2010). Similarly, Leishmania mexicana and Trypanosomecruzi reside and proliferate inside macrophages. Zhang S et al.Delineation of Diverse Macrophage Activation Programs in Response toIntracellular Parasites and Cytokines, PLoS Negl Trop Dis, Vol. 4, No.3: e648 (2010).

Viruses replicate in the host cell, and the accumulation of the viralparticles may result in changes to the plasma membrane. Examples includeHIV, hepatitis C and rhinovirus. Ma Y, et al. NS3 helicase domainsinvolved in infectious intracellular hepatitis C virus particleassembly, J Virol. 82 (15) 7624-39 (2008); Korant B D, Butterworth B E,Inhibition by zinc of rhinovirus protein cleavage: interaction of zincwith capsid polypeptides, J Virol. 18(1):298-306 (1976).

Ultrasound is a technique that may be used to destroy or induceapoptosis of cells. U.S. Pat. No. 6,821,274 (2004). The technique hasbeen used to selectively remove or kill cells based on differences isthe membrane stiffness, such as that caused by cross-linking fromAGE-modification. The ultrasound is targeted to harmonic frequencies ofthe cross-linked cell membranes or components. International PublicationNo. WO2009/143411 (2009).

SUMMARY

In a first aspect, the present invention is a method of treating acondition associate with accumulation of an agent in cells in a patientcomprising exposing the cells to ultrasound, to selectively kill orinduce apoptosis in the cells. The cells include the accumulated agent.The cells may be blood cells, such as red blood cells or white bloodcells.

In a second aspect, the present invention is a method of removing cellsfrom a sample, comprising exposing the sample to ultrasound, toselectively kill or induce apoptosis in the cells. The cells comprise anaccumulated agent.

DETAILED DESCRIPTION

The present invention makes use of the discovery that the differentialresonant frequency of a cell caused by the accumulation of at least oneagent that causes, or is associated with, a pathological or undesiredcondition, such as proteins, lipids, bacteria, viruses, parasites orparticles, may be used to distinguish and eliminate cells in which theaccumulated agent leads to a difference in the resonant frequency of thecell, by applying ultrasound treatment. The cells associated with theaccumulated agent have a resonant frequency which is distinct from cellsof the same type. By selecting the frequency of the ultrasound appliedto the tissue to feed energy into the resonant frequency, the cells withthe accumulated agent will be destroyed or induced to undergo apoptosis.In an aspect of the invention, the cells are not AGE-modified cells. Inanother aspect of the invention, the cells are not tumor cells. In yetanother aspect of the invention, the cells are not cancerous.

The ultrasound technique for removing cell-associated accumulation froma patient is selected for its ability to selectively kill or induceapoptosis in cells having accumulation of the agent associated with thepathological condition, while avoiding removal or destruction of cellsthat do not present the accumulation. For example, cells expressing highlevels of nucleolin on the plasma membrane of the cell may be selecteddue changes in the stiffness and deformability of the cell. As usedherein, “selectively kill or induce apoptosis” means that more of thecells which are the target of the killing or inducing apoptosis are soaffected, as compared to other cell subject to the same exposure.

Ultrasound devices can be used according to practices well known tothose skilled in the art to destroy cells by vibrational techniques, forexample U.S. Pat. No. 5,601,526 (1997) and International Publication No.WO2009/143411 (2009). Ultrasound parameters, such as frequency, powerand pulsation, can be screened for effectiveness in selectivelydestroying the targeted cells. Differential destruction or inducement ofapoptosis may be by selection of the stiffer cells, or by selection ofthe cells by their resonant frequencies. Ultrasound as described abovecan be applied to a subject with monitoring to determine thatinflammatory responses such as fever or swelling do not exceed limitswell known to be safe. This process can be repeated at intervals tomaintain a level of therapeutic benefit. Evaluation of improvement ormaintenance of a desired result can be used to direct the frequency ofreapplication of ultrasound according to the present invention. Theapplication and reapplication can be determined with the goal of gradualimprovement to avoid overwhelming natural mechanisms, such as removal ofcells and cellular debris by scavenging cells.

A variety of techniques are available to determine whether ultrasoundmay be used to selectively remove or kill the cells having theaccumulated agent, that leads to, or is associated with, a pathologicalor undesired condition. The stiffness of individual cells may bedetermined, by techniques such as those described in U.S. Pat. No.6,067,859 (2000). Elastic properties of tissue may be measured, bytechniques such as those described in U.S. Pat. No. 7,751,057 (2010).Furthermore, application of a variety of ultrasound parameters to cellsor a tissue sample, followed by examination of the cells or the tissuesample for destruction or subsequent apoptosis, may also be used todetermine whether ultrasound may selectively remove or kill the cells.

EXAMPLES Example 1 (Prophetic) Ultrasound Removal of Cell-AssociatedAccumulation of Nucleolin in Leukemia Cells

Blood of a patient containing leukemia cells expressing nucleolin on thecell surface is treated with ultrasound. After first diagnosing thepatient, a blood sample is taken for further analysis. Leukemia cellsare isolated from the blood sample, and examined using an opticalstretcher (U.S. Pat. No. 6,067,859), to determine the relative stiffnessof the cells and/or the resonant vibrational frequencies of the cell.This information is then used to select driving frequencies, intensityand length of time of treatment of the ultrasound, to selectivelydestroy or induce apoptosis, in the leukemia cells in vivo or ex vivo.

Example 2 (Prophetic) Ultrasound Removal of Cell-Associated Accumulationof P-Glycoprotein in Colon Cancer Cells

Colon tissue from a patient containing colon cancer cells that present amultidrug resistance (MDR) phenotype are examined and determined to beoverexpressing P-glycoprotein on the cell surface. The cells are thenexamined using an optical stretcher (U.S. Pat. No. 6,067,859), todetermine the relative stiffness of the cells and/or the resonantvibrational frequencies of the cell. This information is then used toselect driving frequencies, intensity and length of time of treatment ofthe ultrasound. For example, an ultrasound generating probe may beincluded at the tip of a colonoscopy device. The ultrasound generatingprobe could generate pulses of ultrasound at the appropriate frequency,to selectively destroy colon cancer cells that present a multidrugresistance (MDR) phenotype, after the probe has been placed proximate tothe tumor. The success of the treatment is monitored by subsequentcolonoscopy.

Example 3 (Prophetic) Ultrasound Removal of Cell-Associated Accumulationof Plasmodium

Blood of a patient, containing erythrocytes infected with Plasmodium, istreated with ultrasound. After first diagnosing the patient, a bloodsample is taken for further analysis. Erythrocytes infected withPlasmodium are isolated from the blood sample, and examined using anoptical stretcher (U.S. Pat. No. 6,067,859), to determine the relativestiffness of the erythrocytes and/or the resonant vibrationalfrequencies of the erythrocytes. In addition, the Plasmodium parasitecould also be examined using the optical stretcher, to determineultrasound parameters capable of direct destruction of the parasite.This information is then used to select driving frequencies, intensityand length of time of treatment of the ultrasound, to selectivelydestroy infected erythrocytes and/or the Plasmodium parasites, in thepatient's blood either in vivo or ex vivo.

Example 4 (Prophetic) Removal of Macrophages and Tubercles Infected withMycobacterium tuberculosis

A biopsy of an area in the lung of a patient containing tubercles andmacrophages infected with M. tuberculosis is taken. The biopsy istreated with ultrasound applied at a range of frequencies andintensities, to determine conditions necessary to selectively destroy orinduce apoptosis in the infected marcophages, the tubercles and/or theM. tuberculosis bacterial cells. The lungs of the patient are thentreated with ultrasound. Time of exposure may range from three to sixtyminutes daily for up to 20 days. At the end of the treatment, thepatients are tested to determine the reduction in the size and/or numberof tubercles present in the lungs of the patient.

Example 5 (Prophetic) Removal of Alginates Caused by Pseudomonasaeruginosa Infection in the Lungs of Patients with Cystic Fibrosis

An alginate sample from the Lungs of the Patient is treated withultrasound applied at a range of frequencies and intensities, todetermine conditions necessary to selectively destroy or break down thealginate. The lungs of the patient are then treated with ultrasound.Time of exposure may range from three to sixty minutes daily for up to20 days. At the end of the treatment, the patient is tested to determinethe reduction of alginates in the lungs.

REFERENCES

-   1. Mi Y, et al. Apoptosis in leukemia cells is accompanied by    alterations in the levels and localization of nucleolin. J Biol Chem    278:8572-9 (2003).-   2. Sven C, et al, Nucleolin Expressed at the Cell Surface is a    Marker of Endothelial Cells in Angiogenic Blood Vessels, Journal of    Cell Biology, Vol. 164, No. 4, 871-878 (2003).-   3. Loo T W, et al. Identification of Residues in the Drug    Translocation Pathway of the Human Multidrug Resistance    P-glycoprotein by Arginine Mutagenesis, Journal of Biological    Chemistry, Vol. 284, No. 36, 24074-24087 (2009).-   4. Brandin P, et al. Prion-Like Transmission of Protein Aggregates    in Neurodegenerative Diseases, Nat Rev Mol Cell Biol. Vol. 11, No.    4, 301-307 (2010).-   5. Perez C, et al. Translational control of cytoplasmic poly A    binding protein (PABP) abundance in HCMV-infected cells, J Virol.    October 27 (2010) Epub.-   6. Persson J, et al. Interleukin-1beta and tumour necrosis    factor-alpha impede neutral lipid turnover in macrophage-derived    foam cells, BMC Immunology, 9(7) (2008).-   7. Vergne I, et al. Cell Biology of Mycobacterium tubercolosis    tuberculosis Phagosome, Ann Rev Cell Dev Biol., Vol. 20, 367-94    (2004).-   8. Moskowitz S M, et al. The Role of Pseudomonas Lipopolysaccharide    in Cystic Fibrosis Airway Infection, Subcell Biochem., Vol. 53,    241-53 (2010).-   9. Hall-Stoodley L, et al., Direct Detection of Bacterial Biofilms    on the Middle-Ear Mucosa of Children With Chronic Otitis Media,    JAMA, Vol. 256, No. 2, 202-11 (2006).-   10. Franke-Fayard B, et al., Sequestration and Tissue Accumulation    of Human Malaria Parasites: Can We Learn Anything from Rodent Models    of Malaria?, PLoS Pathogens, Vol. 6, No. 9, e1001032 (2010).-   11. Zhang S, et al. Delineation of Diverse Macrophage Activation    Programs in Response to Intracellular Parasites and Cytokines, PLoS    Negl Trop Dis, Vol. 4, No. 3: e648 (2010).-   12. Ma Y, et al. NS3 helicase domains involved in infectious    intracellular hepatitis C virus particle assembly, J Virol. 82 (15)    7624-39 (2008).-   13. Korant B D, Butterworth B E, Inhibition by zinc of rhinovirus    protein cleavage: interaction of zinc with capsid polypeptides, J    Virol. 18(1):298-306 (1976).-   14. U.S. Pat. No. 6,821,274 (2004).-   15. International Publication No. WO2009/143411 (2009).-   16. U.S. Pat. No. 5,601,526 (1997).-   17. U.S. Pat. No. 6,067,859 (2000).-   18. U.S. Pat. No. 7,751,057 (2010).

What is claimed is:
 1. A method of treating a condition associated withaccumulation of an agent in cells in a patient, comprising: testing thecells containing the accumulated agent to determine the specificultrasound frequency, power and duration sufficient to selectively killor induce apoptosis in the cells containing the accumulated agent due toa change in the cells due to the accumulated agent, and exposing tissuecontaining the cells of the patient containing the accumulated agent andcontaining cells of the patient without the accumulated agent to thespecific ultrasound frequency, power and duration sufficient toselectively kill or induce apoptosis in the cells containing theaccumulated agent while sparing cells without the accumulated agent dueto differences in stiffness between the cells containing the accumulatedagent and cells without the accumulated agent, wherein the accumulatedagent is a protein, the cells containing the accumulated agent arecancer cells, and the cancer cells have an AGE-modification.
 2. Themethod of claim 1, wherein the testing comprises testing cells takenfrom the patient.
 3. The method of claim 1, wherein the exposingcomprises a plurality of ultrasound treatments.
 4. The method of claim1, wherein the cells containing the accumulated agent are blood cells.5. The method of claim 1, wherein the cells containing the accumulatedagent are white blood cells.
 6. The method of claim 1, wherein thetesting comprises: application of a plurality of ultrasound parametersto cells or a tissue sample, and examination of the cells or the tissuesample for destruction or subsequent apoptosis.
 7. A method of removinga subset of cells from a tissue sample, comprising: testing cells todetermine the specific ultrasound frequency, power and durationsufficient to selectively kill or induce apoptosis in cells containingan accumulated agent due to a change in the cells due to the accumulatedagent, exposing the tissue sample to the specific ultrasound frequency,power and duration sufficient to selectively kill or induce apoptosis inthe subset of cells while sparing cells without the accumulated agentdue to differences in stiffness between cells containing the accumulatedagent and cells without the accumulated agent, wherein the subset ofcells contains the accumulated agent, the tissue sample contains thesubset of cells containing the accumulated agent and contains cellswithout the accumulated agent, the accumulated agent is a protein, thecells containing the accumulated agent are cancer cells, and the cancercells have an AGE-modification.
 8. The method of claim 7, wherein thesample is obtained from a patient.
 9. The method of claim 7, wherein thecells containing the accumulated agent are blood cells.
 10. The methodof claim 7, wherein the cells containing the accumulated agent are whiteblood cells.
 11. The method of claim 7, wherein the testing comprises:application of a plurality of ultrasound parameters to cells or a tissuesample, and examination of the cells or the tissue sample fordestruction or subsequent apoptosis.
 12. The method of claim 7, whereinthe exposing comprises a plurality of ultrasound treatments.